The present invention relates to a plants characterized by an increased content of methionine and methionine related metabolites, to methods of generating same and to uses thereof.
The diets of humans and livestock largely consists of plant material which contains low amounts of several essential amino acids not naturally synthesized by animals or humans. As a result, the nutritional value of plant material and as such plant derived foodstuff is typically limited, oftentimes requiring supplementation of plant derived foodstuff with synthetic amino acids in order to increase it's nutritional value.
Efforts to improve the balance of essential amino acids in the seed proteins of important crops utilizing classical breeding and mutant selection have met with limited success on the laboratory scale and failure on a commercial scale as agronomically acceptable cultivars have not yet been produced.
One of the most important essential amino acid, methionine, exists in limited quantities in legumes, cereals and other crops (Andersen J W., “The Biochemistry of Plants”, Academic Press, NY, 1990, pp. 327-381).
The level of free methionine in plants is very low both in vegetative tissues and seeds. Methionine levels are regulated by both rate of synthesis and metabolism into derivative compounds. Methionine has a short half-life due to rapid conversion to SAM and incorporation into newly synthesized proteins. The incorporation of sulfate, methyl and carbon into methionine and its metabolites has been analyzed in Lemna via tracer elements. It has been observed that methionine is converted into SAM at a rate which is four-folds faster than the incorporation of methionine into proteins (Giovanelli et al., Plant Physiol. 1985, 78:555).
Methionine biosynthesis is subject to regulatory control via cystathionine γ-synthase (CGS), the first enzyme in the Methionine biosynthesis pathway (FIGS. 1a-b). Studies conducted in Lemna have shown that the level of CGS activity decreases in plants grown in the presence of exogenous methionine (Thompson et al., Plant Physiol. 1982, 69:1077). Conversely, treatments that induce methionine deprivation resulted in an increase in the steady-state levels of CGS (Thompson et al., Plant Physiol. 1982, 70:1347).
Thus, it was theorized that methionine may regulate its own synthesis through negative feedback control of cystathionine synthesis. Analysis of Arabidopsis mto1 mutants that over accumulate soluble methionine (Met) revealed that the gene encoding cystathionine gamma-synthase (CGS), the key enzyme in Met biosynthesis, is regulated at the level of mRNA stability and that an amino acid sequence encoded by the first exon of CGS acts in cis to destabilize its own mRNA in a process that is activated by Met or one of its metabolites. The mto1 mutations were shown to be clustered within a small region in the exon, termed mto1, located downstream of the initiator codon (Chiba et al. Science 1999, 286:1371).
Methionine synthesis is also regulated in the biosynthetic pathway of the aspartate family amino acids at the point of competition between threonine synthase (TS) and CGS for their common substrate O-phosphohomoserine (OPH) (FIGS. 1a-b). Evidence for TS-CGS competition and its role in methionine synthesis has been obtained. It has been observed that the mto2-1 mutant of Arabidopsis over-accumulates up to 20-fold more soluble methionine than the wild type plant and displays a marked reduction in threonine levels. The mto2-1 allele carries a point mutation in the TS gene that produces a catalytically impaired enzyme (Bartlem et al. Plant Physiol. 2000, 123:101). Thus, decreased TS activity causes methionine overproduction at the expense of threonine. Furthermore, reducing the level of CGS by antisense methods, which in turn reduce levels of methionine, leads to a seven-fold increase in threonine levels relative to wild-type plants (Kim et al., Plant Science 2000, 151:9). Therefore the levels of both TS and CGS are important in the partitioning of OPH to Met and threonine.
Methionine deficiency in, for example, poultry diets leads to retarded growth, decreased feed conversion efficiency and increased fat content. As such, commercial poultry diets are typically supplemented with synthetic Met (see, for example, U.S. Pat. No. 5,773,052).
In addition, it has been shown that wool growth in sheep and milk production in dairy animals are both limited by the availability of the sulfur amino acids (SAAs) methionine and cysteine (Xu et al., Dairy Sci. 1998, 81:1062).
In attempts to increase the SAA content of seeds, genes encoding SAA-rich proteins from vegetative tissues have been isolated and expressed in seeds under the control of seed-specific regulatory sequences. Such an approach has not resulted in an adequate increase in total target amino acid content in the seed. It has been shown that this and other approaches which attempt to increase SAA content by expression of met-rich proteins are typically limited by the ability of plants to synthesize methionine.
In another approach, genetic engineering methods have been utilized in an attempt to modulate the activity of enzymes catalyzing key steps of relevant biosynthetic pathways (see, for example, European Pat. App. No. 485970).
For example, expression of a mutant form of a bacterial aspartate kinase (AK) which desensitizes negative feed-back inhibition of lysine and threonine production in plants, resulted in a significant overproduction of threonine in vegetative tissue. However, expression of this gene under the control of a seed-specific promoter was shown to raise the levels of methionine content in seeds only three-fold relative to that of wild type plants (Karchi, et al., The Plant J. 1993, 3:721).
Thus, although several approaches have been utilized in efforts to increase the levels of methionine and other essential amino acids in plants, such approaches have failed to produce plants exhibiting a significant increase in methionine levels.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of increasing the content of methionine and its related metabolites in plants, thereby increasing the nutritional and commercial value of such plants.